Nicotine acts as a pharmacological chaperone to up-regulate human α4β2 acetylcholine receptors

Human neuronal nicotinic acetylcholine receptor (AChR) alpha 4 subunits and an alpha 4 mutant (S247F alpha 4) found in autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) were expressed along with beta 2 in permanently transfected tsA201 human embryonic kidney cell lines. Their sensitivity to activation, desensitization, and up-regulation by cholinergic ligands was investigated. Up-regulation after 3 to 24 h resulted primarily from an increase in assembly of AChRs from large pools of unassembled subunits, but up-regulation also resulted from a 5-fold increase in the lifetime of AChRs in the surface membrane. Up-regulation does not require current flow through surface membrane AChRs, because up-regulation occurs in the presence of the channel blocker mecamylamine and with the alpha 4 mutant, which prevents nearly all AChR function. Both membranepermeable ligands like nicotine and much less permeable quaternary amine cholinergic ligands can act as pharmacological chaperones within the endoplasmatic reticulum to promote the assembly of AChRs. Agonists are more potent pharmacological chaperones than antagonists, presumably because activated or desensitized conformations assemble more efficiently. Assembly intermediates are disrupted by solubilization in Triton X-100, but chemical cross-linking stabilizes a putative assembly intermediate approximately the size of an alpha 4 beta 2 alpha 4 beta 2 tetramer.